Our laboratory takes a combination of genetic, biochemical, and visual approaches to the study of intracellular lipid trafficking in the yeast S. cerevisiae. Little is known about how the lipid composition of organelles is established and maintained or how lipids are trafficked in cells. The lipid composition of organelles is thought to be important for their proper function and for maintaining organelle identity. In addition, many diseases are associated with disregulations of intracellular lipid trafficking. Sterol distribution between the ER and plasma membrane. Cells maintain a sterol gradient across the secretory system, with the lowest concentrations in the ER and the highest in the plasma membrane (PM). Little is known about how this heterogeneous distribution of cholesterol is maintained despite continuous vesicular traffic between organelles. Both the modulation of the sterol content of transport vesicles and the non-vesicular transport of cholesterol between organelles are likely to contribute. We have found that sterol transport from the PM to the ER is nonvesicular and ATP-independent. It is likely that this transport pathway is nonvectorial and can shuttle sterol between the PM and ER. Therefore, it probably cannot maintain the sterol gradient between the ER and PM by itself. Rather we find that intracellular sterol distribution is largely determined by the affinity of sterols for sterol-sphingolipid-enriched microdomains (rafts), which are highly enriched in the PM. Exogenous sterols with low affinity for rafts are distributed differently within the cell than sterols with higher affinity. We propose that the concentration of non-raft associated sterol in the PM and ER are similar and that raft association in the PM drives the difference in the sterol concentration in the PM and ER. Raft-association is likely to be important for sterol distribution throughout the cell. We have submitted a paper describing these results. In addition, we are currently examining if raft-association is also important for determining the intracellular distribution of sphingolipids, which is similar to that of sterols. Lipid droplet biogenesis and function. Lipid droplets are found in almost all eukaryotic cells and are important for neutral lipid storage and lipid homeostasis. Very little is known about how these structures form or how their lipid content is regulated. We have isolated mutants with defects in lipid droplet assembly and are in the process of characterizing them. These mutants appear to have defects primarily in proteins that regulate the neutral lipid content of the cell, but some may also lack proteins required for lipid droplet biogenesis. In addition, we have constructed a strain that cannot synthesize neutral lipid. We have found that this strain is hypersensitive to some exogenous lipids and will be a good model for studying lipotoxicity, which is important for the etiology of many diseases.